Experimental multivisceral xenotransplantation

Background: Organ shortage impairs the proposition of multivisceral transplantation to treat multiple organ failure. Interspecies (xeno) transplantation is a valid solution for organ shortage; however, suitable models of this advance are lacking. We describe an effective model of multivisceral xenotransplantation to study hyperacute rejection. Methods: Under general anesthesia, we in block recovered the distal esophagus, stomach, small bowel, colon, liver, pancreas, spleen, and kidneys from donors and implanted heterotopically in the lower abdomen of recipients. Animals were divided into four groups: I-canine donor, swine recipient (n = 6); II - swine donor, canine recipient (n = 5); III-canine donor, canine recipient (n = 4); and IV-swine donor, swine recipient (n = 5). Groups I and 11 comprised experimental (xenotransplantation) and III and IV control groups (allotransplantation). During the experiment, we appraised recipient evolution and graft modification by sequential biopsy up to 3 h. At this time, we killed animals for autopsy (experimental end point). Results: We accomplished all experiments successfully. Every grafts attained customary appearance and convenient urine output immediately after unclamp. Around 15 min after reperfusion, xenografts achieved signs of progressive hyperacute rejection and absence of urine output. At the end of experiments we observed moderate to severe hyperacute rejection at small bowel, colon, mesenteric lymph node, liver, spleen, pancreas, and kidney, while stomach and esophagus achieved mild lesions. In contrast, allograft achieved normal or minimum ischemia/reperfusion injury and constant urine output. Conclusion: The present procedure assembles a simple and effective model to study multivisceral xenotransplantation and may ultimately spread researches toward hyperacute rejection.

Establishment and characterization of androgen-independent human prostate cancer cell lines, PcBra1, PcBra2, and PcBra3

One of the main obstacles for understanding biological events involved in cancer is the lack of experimental models for in vitro studies especially for prostate cancer (PC).There are a limited number of PC cell lines being the majority originated from metastatic tumors mostly acquired from American Tissue Cell Culture which demands importation an expensive and bureaucratic process. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors and the research based on cell lines derived from Brazilians should be interesting. Our aim was to develop tumor cell lines from primary PC.

DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin

Resistance to chemotherapeutic drugs can be an obstacle to a successful treatment of cancer patients in part associated with individual response and differences in the DNA repair system. The Comet assay is an informative test to investigate DNA damage and repair in cells in response to a variety of DNA-damaging agents, including chemotherapeutic drugs. The aim of this study was to assess leukocytes damage after in-vitro cisplatin treatment and DNA repair action using the Comet assay in 20 patients with melanoma and 20 cancer-free individuals. Leukocytes` DNA damage before and after cisplatin treatment, in three different concentrations, was analyzed. The DNA repair capability was investigated after 1-5 h of in-vitro cells growing without cisplatin. The Comet score of the patients` basal DNA damage was higher than that observed in controls, but the difference was not statistically significant (P=0.85). Although both groups had similar Comet scores to all cisplatin concentrations tested and the DNA repair times, the basal DNA damage (P < 0.001) and cisplatin damages (P < 0.005) were statistically lower than the different repair times investigated. Considering the progressive increase in the Comet score due to repair time, the negative results here observed could be associated with the reduced cell culture incubation that should be better evaluated. Considering the mutagenic action of cisplatin on tumor cells and the importance of individual DNA repair mechanisms in the chemotherapeutic melanoma treatment, the peripheral leukocytes could be particularly useful as a tool for DNA repair response identified by the Comet assay. Melanoma Res 21:99-105 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Influence of Suture on Peripheral Nerve Regeneration and Collagen Production at the Site of Neurorrhaphy: An Experimental Study

BACKGROUND: Restoration of nerve continuity and effective maintenance of coaptation are considered fundamental principles of end-to-end peripheral nerve repair. OBJECTIVE: To evaluate the influence of the number of stitches on axonal regeneration and collagen production after neurorrhaphy. METHODS: Thirty male Wistar rats were equally divided into 3 groups and were all operated on with the right sciatic nerve exposed. In 2 groups, the nerve was sectioned and repaired by means of 3 (group B) or 6 (group C) epineurium sutures with 100 monofilament nylon. One group (group A) was used as a control. Each animal from groups B and C underwent electrophysiological evaluation with motor action potential recordings before nerve section and again at an 8-week interval after neurorrhaphy. Nerve biopsy specimens were used for histomorphometric assessment of axonal regeneration and quantification of collagen at the repair site. RESULTS: Animals from group C had significantly lower motor action potential conduction velocities compared with control animals (P = .02), and no significant difference was seen between groups B and C. Parameters obtained from morphometric evaluation were not significantly different between these 2 groups. Type I collagen and III collagen in the epineurium were significantly higher in group C than in either the control group (P = .001 and P = .003) or group B (P = .01 and P = .02). No differences were identified for collagen I and III in the endoneurium. CONCLUSION: Using 6 sutures for nerve repair is associated with worse electrophysiological outcomes and higher amounts of type I and III collagen in the epineurium compared with control. Neurorraphy with 6 stitches is also related to a significant increase in epineurium collagen I and III compared with 3-stitch neurorraphy.

Markers of vascular differentiation, proliferation and tissue remodeling in juvenile nasopharyngeal angiofibromas

Juvenile nasopharingeal angiofibroma (JNA) is a histologically benign locally aggressive tumor characterized by irregular vessels embedded. in a fibrous stroma. Excessive vascularity results in bleeding complications, and the inhibition of angiogenesis is a promising strategy for managing extensive JNA tumors. To better characterize the endothelial components of JNA, we aimed to evaluate markers of vascular differentiation and proliferation, such as friend leukemia integration-1 (FLI-1) and endoglin, lymphatic markers, including podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3) and its cognate ligand VEGFC, GLUT-1, a diagnostic marker that discriminates between hemangiomas and vascular malformations, and two markers of tissue remodeling, stromelysin 3 (ST3) and secreted acid protein rich in cysteine (SPARC). Antigens were assessed immunohistochemically in vessels and stromal cells of JNA archival cases (n=22). JNA endothelial cells were positive for endoglin, VEGFC and FLI-1, whereas podoplanin and VEGFR3 were negative in all cases. Both endothelial cells and fibroblasts stained for ST3 and SPARC. GLUT-1 was investigated in JNA cases, in infantile hemangiomas (n=123) and in vascular malformations (n=135) as controls. JNAs and vascular malformations were GLUT-1-negative, while hemangiomas showed positive staining. The presence of markers of endothelial differentiation and proliferation highlighted the hyper-proliferative state of JNA vessels. The absence of podoplanin and VEGFR3 underscores their blood endothelial cell characteristic. The absence of GLUT-1 discriminates JNAs from hemangiomas. ST3 and SPARC up-regulation in endothelial cells and fibroblasts may contribute to a compensatory signaling for controlling angiogenesis. Some of these markers may eventually serve as therapeutic targets. Our results may aid in the understanding of JNA pathophysiology.

Histologic Study of Perifascial Areolar Tissue Implanted in Rabbit Vocal Folds: An Experimental Study

Objectives: Perifascial areolar tissue (PAT) consists of loose areolar tissue with viscoelastic properties that are similar to those found in tissues in the superficial layer of the vocal fold. The aim of this study was to quantify the inflammatory process and the collagen content of the graft, as well as that of the host tissue, after placement of a strip of PAT into the rabbit vocal fold. Methods: Surgeries were performed on 30 rabbits. The grafts were implanted in pockets that were surgically created in the right vocal fold. The left vocal fold (control group) was subjected only to surgical manipulation. The animals were divided into 3 groups for evaluations at 15 days, 3 months, and 6 months, and their larynx tissues were subsequently reviewed by histology. Results: The grafts were characterized by disorganized and thick collagen bundles and were identified in all study groups. The collagen density stayed constant over time. There was an acute inflammatory response induced by the graft at 15 clays that did not exist in the specimens taken at 3 and 6 months. Deposition of collagen fibers in the lamina propria was observed starting at 15 days after the operation and was more intense in the experimental vocal fold than in the control vocal fold. Conclusions: Our findings indicated that PAT has a low tendency for promoting an inflammatory response. However, there was a loss of the original architecture of the graft tissue and a greater deposition of collagen in the implanted vocal folds than in the control group.

Potentializing the effects of GM1 by hyperbaric oxygen therapy in acute experimental spinal cord lesion in rats

Study design: Experimental, controlled, animal study. Objectives: To evaluate the effect of GM1 ganglioside, hyperbaric oxygen and both in combination, in the treatment of experimental spinal cord lesions in rats. Setting: Brazil. Methods: Thirty-two Wistar rats with spinal cord lesions were divided into four groups: one group received GM1 ganglioside, one was submitted to hyperbaric oxygen therapy (HBOT), the third received both treatments and the fourth received no treatment (control). Results: There were no significant differences between the groups in the histological analysis, for any of the variables (necrosis, hemorrhage, hyperemia, cystic degeneration, P>0.06). Neither were there any significant differences in the comparison of left and right sides in the functional tests (P>0.06 for all). No significant differences were found in the locomotor ratings, in the comparison of groups at 2, 7, 21 and 28 days after the surgical procedure. However, in the evaluation on day 14, group 3, which received the combined therapy, showed a significantly higher Basso Beattie and Bresnahan score than the other groups (P = 0.015). Conclusion: The therapeutic effect of GM1 in locomotor evaluation of rats submitted to spinal cord lesion is anticipated by HBOT. Spinal Cord (2010) 48, 808-813; doi:10.1038/sc.2010.37; published online 27 April 2010

Prone position prevents regional alveolar hyperinflation and mechanical stress and strain in mild experimental acute lung injury

Prone position may delay the development of ventilator-induced lung injury (VILI), but the mechanisms require better elucidation. In experimental mild acute lung injury (ALI), arterial oxygen partial pressure (Pa(O2)), lung mechanics and histology, inflammatory markers [interleukin (IL)-6 and IL-1 beta], and type III procollagen (PCIII) mRNA expressions were analysed in supine and prone position. Wistar rats were randomly divided into two groups. In controls, saline was intraperitoneally injected while ALI was induced by paraquat. After 24-h, the animals were mechanically ventilated for 1-h in supine or prone positions. In ALI, prone position led to a better blood flow/tissue ratio both in ventral and dorsal regions and was associated with a more homogeneous distribution of alveolar aeration/tissue ratio reducing lung static elastance and viscoelastic pressure, and increasing end-expiratory lung volume and Pa(O2). PCIII expression was higher in the ventral than dorsal region in supine position, with no regional changes in inflammatory markers. In conclusion, prone position may protect the lungs against VILI, thus reducing pulmonary stress and strain. (C) 2009 Elsevier B.V. All rights reserved.

Pulmonary lesion induced by low and high positive end-expiratory pressure levels during protective ventilation in experimental acute lung injury

Objective: To investigate the effects of low and high levels of positive end-expiratory pressure (PEEP), without recruitment maneuvers, during lung protective ventilation in an experimental model of acute lung injury (ALI). Design: Prospective, randomized, and controlled experimental study. Setting: University research laboratory. Subjects: Wistar rats were randomly assigned to control (C) [saline (0.1 ml), intraperitoneally] and ALI [paraquat (15 mg/kg), intra peritoneally] groups. Measurements and Main Results: After 24 hours, each group was further randomized into four groups (six rats each) at different PEEP levels = 1.5, 3, 4.5, or 6 cm H(2)O and ventilated with a constant tidal volume (6 mL/kg) and open thorax. Lung mechanics [static elastance (Est, L) and viscoelastic pressure (Delta P2, L)] and arterial blood gases were measured before (Pre) and at the end of 1-hour mechanical ventilation (Post). Pulmonary histology (light and electron microscopy) and type III procollagen (PCIII) messenger RNA (mRNA) expression were measured after 1 hour of mechanical ventilation. In ALI group, low and high PEEP levels induced a greater percentage of increase in Est, L (44% and 50%) and Delta P2, L (56% and 36%) in Post values related to Pre. Low PEEP yielded alveolar collapse whereas high PEEP caused overdistension and atelectasis, with both levels worsening oxygenation and increasing PCIII mRNA expression. Conclusions: In the present nonrecruited ALI model, protective mechanical ventilation with lower and higher PEEP levels than required for better oxygenation increased Est, L and Delta P2, L, the amount of atelectasis, and PCIII mRNA expression. PEEP selection titrated for a minimum elastance and maximum oxygenation may prevent lung injury while deviation from these settings may be harmful. (Crit Care Med 2009; 37:1011-1017)

POLYETHYLENE GLYCOL ADDITION DOES NOT IMPROVE EXOGENOUS SURFACTANT FUNCTION IN AN EXPERIMENTAL MODEL OF MECONIUM ASPIRATION SYNDROME

Meconium (MEC) is a potent inactivator of pulmonary surfactant. The authors studied the effects of polyethylene glycol addition to the exogenous surfactant over the lung mechanics and volumes. Human meconium was administrated to newborn rabbits. Animals were ventilated for 20 minutes and dynamic compliance, ventilatory pressure, and tidal volume were recorded. Animals were randomized into 3 study groups: MEC group (without surfactant therapy); S100 group (100 mg/kg surfactant); and PEG group (100 mg/kg porcine surfactant plus 5% PEG). After ventilation, a pulmonary pressure-volume curve was built. Histological analysis was carried out to calculate the mean alveolar size (Lm) and the distortion index (DI). Both groups treated with surfactant showed higher values of dynamic pulmonary compliance and lower ventilatory pressure, compared with the MEC group (P .05). S100 group had a larger maximum lung volume, V30, compared with the MEC group (P .05). Lm and DI values were smaller in the groups treated with surfactant than in the MEC group (P .05). No differences were observed between the S100 and PEG groups. Animals treated with surfactant showed significant improvement in pulmonary function as compared to nontreated animals. PEG added to exogenous surfactant did not improve lung mechanics or volumes.

Intravenous glutamine decreases lung and distal organ injury in an experimental model of abdominal sepsis

Introduction The protective effect of glutamine, as a pharmacological agent against lung injury, has been reported in experimental sepsis; however, its efficacy at improving oxygenation and lung mechanics, attenuating diaphragm and distal organ injury has to be better elucidated. In the present study, we tested the hypothesis that a single early intravenous dose of glutamine was associated not only with the improvement of lung morpho-function, but also the reduction of the inflammatory process and epithelial cell apoptosis in kidney, liver, and intestine villi. Methods Seventy-two Wistar rats were randomly assigned into four groups. Sepsis was induced by cecal ligation and puncture surgery (CLP), while a sham operated group was used as control (C). One hour after surgery, C and CLP groups were further randomized into subgroups receiving intravenous saline (1 ml, SAL) or glutamine (0.75 g/kg, Gln). At 48 hours, animals were anesthetized, and the following parameters were measured: arterial oxygenation, pulmonary mechanics, and diaphragm, lung, kidney, liver, and small intestine villi histology. At 18 and 48 hours, Cytokine-Induced Neutrophil Chemoattractant (CINC)-1, interleukin (IL)-6 and 10 were quantified in bronchoalveolar and peritoneal lavage fluids (BALF and PLF, respectively). Results CLP induced: a) deterioration of lung mechanics and gas exchange; b) ultrastructural changes of lung parenchyma and diaphragm; and c) lung and distal organ epithelial cell apoptosis. Glutamine improved survival rate, oxygenation and lung mechanics, minimized pulmonary and diaphragmatic changes, attenuating lung and distal organ epithelial cell apoptosis. Glutamine increased IL-10 in peritoneal lavage fluid at 18 hours and bronchoalveolar lavage fluid at 48 hours, but decreased CINC-1 and IL-6 in BALF and PLF only at 18 hours. Conclusions In an experimental model of abdominal sepsis, a single intravenous dose of glutamine administered after sepsis induction may modulate the inflammatory process reducing not only the risk of lung injury, but also distal organ impairment. These results suggest that intravenous glutamine may be a potentially beneficial therapy for abdominal sepsis.

Methylprednisolone improves lung mechanics and reduces the inflammatory response in pulmonary but not in extrapulmonary mild acute lung injury in mice

Objective: Gorticosteroids have been proposed to be effective in modulating the inflammatory response and pulmonary tissue remodeling in acute lung injury (ALI). We hypothesized that steroid treatment might act differently in models of pulmonary (p) or extrapulmonary (exp) ALI with similar mechanical compromise. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: One hundred twenty-eight BALB/c mice (20-25 g). Interventions: Mice were divided into six groups. In control animals sterile saline solution was intratracheally (0.05 mL, Cp) or intraperitoneally (0.5 mL, Gexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (10 mu g, ALIp) or intraperitoneally (125 mu g, ALIexp). Six hours after lipopolysaccharide administration, ALIp and ALlexp animals were further randomized into subgroups receiving saline (0.1 mL intravenously) or methylprednisolone (2 mg/kg intravenously, Mp and Mexp, respectively). Measurements and Main Results: At 24 hrs, lung state elastance, resistive and viscoelastic pressures, lung morphometry, and collagen fiber content were similar in both ALI groups. KC, interieukin-6, and transforming growth factor (TGF)-beta levels in bronchoatveolar lavage fluid, as well as tumor necrosis factor (TNF)-alpha, migration inhibitory factor (MIF), interferon (IFN)-gamma, TGF-beta 1 and TGF-beta 2 messenger RNA expression in lung tissue were higher in ALIp than in ALIexp animals. Methylprednisolone attenuated mechanical and morphometric changes, cytokine levels, and TNF-alpha, MIF, IFN gamma, and TGF-beta 2 messenger RNA expression only in ALIp animals, but prevented any changes in collagen fiber content in both ALI groups. Conclusions. Methylprednisolone is effective to inhibit fibrogenesis independent of the etiology of ALI, but its ability to attenuate inflammatory responses and lung mechanical changes varies according to the cause of ALI.

Recruitment maneuver in pulmonary and extrapulmonary experimental acute lung injury

Objective. The aim of this study is to test the hypothesis that recruitment maneuvers (RMs) might act differently in models of pulmonary (p) and extrapulmonary (exp) acute lung injury (ALI) with similar transpulmonary pressure changes. Design: Prospective, randomized, controlled experimental study. Setting. University research laboratory. Subjects: Wistar rats were randomly divided into four groups. In control groups, sterile saline solution was intratracheally (0.1 mL, Cp) or intraperitoneally (1 mL, Cexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (100 jig, ALIp) or intraperitoneally (1 mg, ALIexp). After 24 hrs, animals were mechanically ventilated (tidal volume, 6 mL/kg; positive end-expiratory pressure, 5 cm H2O) and three RMs (pressure inflations to 40 cm H2O for 40 secs, 1 min apart) applied. Measurements and Main Results. Pao(2), lung resistive and viscoelastic pressures, static elastance, lung histology (light and electron microscopy), and type III procollagen messenger RNA expression in pulmonary tissue were measured before RMs and at the end of 1 hr of mechanical ventilation. Mechanical variables, gas exchange, and the fraction of area of alveolar collapse were similar in both ALI groups. After RMs, lung resistive and viscoelastic pressures and static elastance decreased more in ALIexp (255%,180%, and 118%, respectively) than in ALIp (103%, 59%, and 89%, respectively). The amount of atelectasis decreased more in ALIexp than in ALIp (from 58% to 19% and from 59% to 33%, respectively). RMs augmented type III procollagen messenger RNA expression only in the ALIp group (19%), associated with worsening in alveolar epithelium injury but no capillary endothelium lesion, whereas the ALIexp group showed a minor detachment of the alveolar capillary membrane. Conclusions. Given the same transpulmonary pressures, RMs are more effective at opening collapsed alveoli in ALIexp than in ALIp, thus improving lung mechanics and oxygenation with limited damage to alveolar epithelium.

Impact of pressure profile and duration of recruitment maneuvers on morphofunctional and biochemical variables in experimental lung injury

Objective: To investigate the effects of the rate of airway pressure increase and duration of recruitment maneuvers on lung function and activation of inflammation, fibrogenesis, and apoptosis in experimental acute lung injury. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: Thirty-five Wistar rats submitted to acute lung injury induced by cecal ligation and puncture. Interventions: After 48 hrs, animals were randomly distributed into five groups (seven animals each): 1) nonrecruited (NR); 2) recruitment maneuvers (RMs) with continuous positive airway pressure (CPAP) for 15 secs (CPAP15); 3) RMs with CPAP for 30 secs (CPAP30); 4) RMs with stepwise increase in airway pressure (STEP) to targeted maximum within 15 secs (STEP15); and 5) RMs with STEP within 30 secs (STEP30). To perform STEP RMs, the ventilator was switched to a CPAP mode and positive end-expiratory pressure level was increased stepwise. At each step, airway pressure was held constant. RMs were targeted to 30 cm H(2)O. Animals were then ventilated for 1 hr with tidal volume of 6 mL/kg and positive end-expiratory pressure of 5 cm H(2)O. Measurements and Main Results: Blood gases, lung mechanics, histology (light and electronic microscopy), interleukin-6, caspase 3, and type 3 procollagen mRNA expressions in lung tissue. All RMs improved oxygenation and lung static elastance and reduced alveolar collapse compared to NR. STEP30 resulted in optimal performance, with: 1) improved lung static elastance vs. NR, CPAP15, and STEP15; 2) reduced alveolar-capillary membrane detachment and type 2 epithelial and endothelial cell injury scores vs. CPAP15 (p < .05); and 3) reduced gene expression of interleukin-6, type 3 procollagen, and caspase 3 in lung tissue vs. other RMs. Conclusions: Longer-duration RMs with slower airway pressure increase efficiently improved lung function, while minimizing the biological impact on lungs. (Crit Care Med 2011; 39:1074-1081)

Early and late effects of bone marrow-derived mononuclear cell therapy on lung and distal organs in experimental sepsis

We tested the hypothesis that bone marrow-derived mononuclear cells (BMDMCs) at an early phase of cecal ligation and puncture (CLP)-induced sepsis may have lasting effects on: (1) lung mechanics and histology, (2) the structural remodelling of lung parenchyma, (3) lung, kidney, and liver cell apoptosis, and (4) pro- and anti-inflammatory cytokines and growth factors. At day 1, BMDMC significantly reduced mortality, as well as caspase-3, interleukin (IL)-6 and IL-1 beta vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, and transforming growth factor-beta, but increased IL-10 mRNA expression in lung tissue in septic mice contributing to endothelium and epithelium alveolar repair and improvement of lung mechanics. BMDMC also prevented the increase of apoptotic cells in lung, liver, and kidney. At day 7, these early functional and morphological effects were preserved or further improved. In conclusion, in the present model of sepsis, the beneficial effects of early administration of BMDMCs on lung and distal organs were preserved, possibly by paracrine mechanisms. (C) 2011 Elsevier B.V. All rights reserved.